By independent segregation of the chromosomes, selectable marker-free transgenic rice with the transgene only can be produced

By independent segregation of the chromosomes, selectable marker-free transgenic rice with the transgene only can be produced. OD reading are 0.590 0.023 and 0.574 0.019, em p /em = 0.119. Data are shown as means SD. 1472-6750-11-37-S2.TIFF (2.3M) GUID:?DBE5E82E-7AC2-4F8A-8457-3291209B45C6 Abstract Background Human insulin-like growth factor-I (hIGF-I) is a growth factor which is highly resemble to insulin. It is essential for cell proliferation and has been proposed for treatment of various endocrine-associated diseases including growth hormone insensitivity syndrome and diabetes mellitus. In the present study, an efficient herb expression system was developed to produce biologically active recombinant hIGF-I (rhIGF-I) in transgenic rice grains. Results The plant-codon-optimized hIGF-I was introduced into rice via em Agrobacterium /em -mediated transformation. To enhance the stability and yield of rhIGF-I, the endoplasmic reticulum-retention signal and glutelin signal peptide were used to deliver rhIGF-I to endoplasmic reticulum for stable accumulation. We found that only glutelin signal peptide could lead to successful expression of hIGF-I and one gram of hIGF-I rice grain possessed the maximum activity level equivalent to 3.2 micro molar of commercial rhIGF-I. em In vitro /em functional analysis showed that this rice-derived rhIGF-I was effective in inducing membrane ruffling and glucose uptake on rat skeletal muscle cells. Oral meal test with rice-containing rhIGF-I acutely reduced blood glucose levels in streptozotocin-induced and Zucker diabetic rats, whereas it had no effect in normal rats. Conclusion Our findings provided an alternative expression system to produce large quantities of biologically active rhIGF-I. The provision of large quantity of recombinant proteins will promote further research around the therapeutic potential of rhIGF-I. strong class=”kwd-title” Keywords: Oryza sativa L., herb bioreactor; transgenic herb; recombinant protein; protein targeting; KDEL; IGF-I Background Human insulin-like growth factor-I (hIGF-I) plays a critical role in cellular differentiation, proliferation, growth and apoptosis [1]. It is a single polypeptide chain of 70 amino acid residues and is encoded by a single gene on chromosome 12, with a molecular weight of 7646 Da [2,3]. It has 50% amino acid sequence homology with insulin. Hence, hIGF-I has been proposed as an alternative therapeutic agent to treat diabetic mellitus, especially for those patients with defects in insulin receptors. Apart from diabetes mellitus, the efficacy of hIGF-I in the treatment of growth disorders as well as insulin resistance has been studied [4]. In late 2005, recombinant hIGF-I Rabbit Polyclonal to RBM16 (rhIGF-I) was approved by the United States Food and Drug Administration (USFDA) as a therapeutic agent for the growth hormone insensitivity syndrome (GHIS) [5]. Recombinant hIGF-I was first synthesized by recombinant DNA techniques in 1986. To date, commercial rhIGF-I is mainly produced in different organisms [6-8]. Problems of these processes include low expression levels, high gear and production costs, incorrect post-translational modifications as well as potential contamination with human pathogens. With the advance in genetic engineering, rice is recently recognized as a promising alternative for the production of safe and economical biopharmaceutical in large quantities [9]. Rice offers the advantage of producing large quantity of proteins in terms of cost, product safety, scalability and authenticity [9]. Rice is known to exclude any noxious chemicals such as nicotine and toxic alkaloids in tobacco as well as having low allergenicity. Large amount of recombinant proteins can be synthesized at one time as a single rice herb can produce 3-Methylcrotonyl Glycine over 1,000 grains. Besides, as rice is self-pollinated, simple regulatory rules like isolation distances can be set up to prevent cross-pollinating and out crossing of the transgenic trait. In an attempt to achieve high-yield expression of foreign genes in plants, the coding sequence of heterologous gene has to be altered to plant-preferred codons. Previous studies have showed that codon usage biases are strongly correlated with gene expression levels [10]. Highly expressed genes preferentially use a subset of “optimal” codons which correspond to the most abundant tRNAs, leading to enhanced translation accuracy and efficiency [11,12]. Moreover, proteins yields can be increased if the protein is directed to specific compartments in order to prevent degradation by the proteolytic system of the cells. In a herb cell (as in all eukaryotic cells), an amino-terminal signal peptide can direct proteins to the secretory pathway, including the endoplasmic reticulum (ER), the Golgi complex and hydrolytic compartments (vacuoles in plants), or to secretion from the cell. It has been found that secretory proteins 3-Methylcrotonyl Glycine could be accumulated to high level than those expressed in cytosol [13]. Some studies found that proteins yields can be further enhanced if the protein is retained in the ER lumen using the Lys-Asp-Glu-Leu (KDEL) C-terminal tetrapeptide 3-Methylcrotonyl Glycine [14]. Protein levels were 6-14 occasions higher in cells transformed with the construct made up of KDEL than that without KDEL [15]. In the present study, we hypothesized that 3-Methylcrotonyl Glycine functional rhIGF-I could be produced in transgenic rice grains. Codons of cDNA of hIGF-I was altered.